(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Endpoint summary

Description of key information

Skin sensitisation.OECD 442C: Solutions of MEDOL-10 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. The overall depletion value of 65.6% places MEDOL-10 in the reactivity class of “high” and hence it is predicted by DPRA to be a skin sensitizer.

Skin sensitisation.OECD 442E: The test item MEDOL-10 with a log Pow of 1.92 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Skin sensitisation.QSAR TOPKAT: TOPKAT predicted MEDOL-10 to be sensitizer. The predicted structure is within applicability domain, therefore this positive result may be regarded as moderate reliable.

Skin sensitisation.QSAR CAESAR: CAESAR predicted MEDOL-10 to be sensitizer. According to VEGA’s evaluation scheme, the prediction may be not reliable. The uncertainty in the prediction included moderate concordance, moderate accuracy and one unknown fragment in the training set. In addition,
MEDOL-10 was outside applicability domain of the model. As the average similarity of the structural analogues is still high, the prediction may be assessed as low reliable for a weight of- evidence approach.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 30 November 2017
Experimental completion date: 13 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct; 26(7):1150-60.

Qualifier:

equivalent or similar to guideline

Guideline:

other: A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug; 38(4):275-84.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Identification: MEDOL-10
Chemical Name: (2-Ethyl-2-methyl-1,3-dioxolan-4-yl)methyl acrylate
CAS No.: 69701-99-1
Batch: 09892701
Purity: 99.9%
Partition coefficient (n-octanol/water): log Pow: 1.92
Water solubility: 12.92 g/L
Appearance: Colourless liquid
Expiry Date: 06 March 2019
Storage Conditions: At room temperature, protected from light
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Test Item Preparation

The maximum concentration of test item was 5000 μg/mL stable suspended in culture medium), as tested by a solubility test.

For the first XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 μg/mL culture medium.
Due to strong cytoxicity observed in all test item treated cells, the first XTT test was repeated with adjusted test item concentrations, starting with 50 μg/mL in culture medium as the highest concentration.

On the day of the experiment (immediately prior to start) MEDOL-10 was dissolved in culture medium.

Test Systems and Supporting Information

Reasons for the Choice of THP-1 Cells

THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures

Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank (aliquots of cells in freezing medium at 1 × 10 6 to 2 × 10 6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10 6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).

The passage numbers of the used THP-1 cells were 26 in both XTT assays and 18 and 11 in the h-CLAT for runs 1 and 2, respectively.

Culture Medium

RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

Preparation and Seeding of THP-1 Cells

On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10 6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.

For the main experiment (h-CLAT) 0.9 - 1 × 10 6 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.

Experimental Design and Procedures of XTT

Dose Finding Assay (XTT Test)

The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.

The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria.

XTT Labelling Mixture

The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.

Treatment

After the cell seeding, 100 μL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.

XTT Labelling and Measurement

At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).


Calculation of the h-CLAT Test Item Concentrations

Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).

Eight final concentrations (μg/mL) were used for the test item in the main experiment (h-CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Acceptability of the Assay

The XTT test is considered to be acceptable if it meets the following criteria:

• mean absorbance of the medium control is ≥ 0.5

• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Experimental Design and Procedures of h-CLAT

The test item was tested in two independent runs.

Treatment of the Cells

For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.

Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.

Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells

The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).

All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.

The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement

After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Flow Cytometry Acquisition

Before using the flow cytometer (FACSCalibur) the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.

The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition

The following acquisition plots were prepared:

• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)

• Histogram plot of each channel (FL-1 and FL-3, respectively)

The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells.

The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition

Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

For full details on Data Analysis and Interpretation, please refer to entry in "Any other information on materials and methods incl. tables" section.

Test Item Concentrations

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):
13.9, 16.7, 20.0, 24.0, 28.8, 34.6, 41.5 and 49.8 μg/mL

The highest test item concentration for the main experiment (h-CLAT) of MEDOL-10 was previously determined by two XTT tests.
Cytotoxic effects were observed following incubation with the highest tested concentration (50 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 41.5 μg/mL.

Vehicle / solvent control:

DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Remarks:

Value is a mean of both XTT tests. CV75 values were 43.4 and 39.7 ug/mL respectively for the first and second XTT tests

Value:

41.5 µg/mL

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI, CD 54 >/= 200 %

Value:

150

Cell viability:

> 50%

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Parameter:

other: RFI

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

The test item with a log Pow of 1.92 was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Results of the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item MEDOL-10

The mean viability of the solvent control in comparison to the medium control was 107.35%. The CV75 value of the first XTT test: 43.3 μg/mL.

For tabulated results please refer to attachment "Results of the first and second XTT tests for Test Item MEDOL-10".

Results of the second XTT test for Test Item MEDOL-10

The mean viability of the solvent control in comparison to the medium control was 91.17%.

The CV75 value of the second XTT test: 39.7 μg/mL
The mean CV75 value of both XTT tests: 41.5 μg/mL

For tabulated results please refer to attachment "Results of the first and second XTT tests for Test Item MEDOL-10".

Results of the h-CLAT Test

For results of the first and second h-CLAT run for the Test Item MEDOL-10 please refer to attachments "Results of the first and second h-CLAT runs for the Test Item MEDOL-10".

 

Conclusions:

The test item MEDOL-10 with a log Pow of 1.92 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

Introduction-

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of MEDOL-10 dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of MEDOL-10 was previously determined by two XTT tests.

Results-

Cytotoxic effects were observed following incubation with the highest tested concentration (50 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 41.5 μg/mL.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Conclusion-

In conclusion, the test item MEDOL-10 with a log Pow of 1.92 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 12 January 2018
Experimental completion date: 02 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Sponsor’s identification: MEDOL-10
Chemical name: (2-Ethyl-2-methyl-1,3-dioxolan-4-yl) methyl acrylate
CAS No.: 69701-99-1
Batch No.: 09892701
Purity/correction factor: 99.9%
Molecular weight: 200.23 g/moL
Appearance: Colourless liquid
Expiry/retest date: 27 February 2018
Storage conditions: Ambient temperature (15°C to 25°C) in the dark

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Assessment of Test Item Solubility

The solubility of MEDOL-10 in was assessed at a concentration of 100 mM in acetonitrile.

Preparation of Peptide Stock Solutions

Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards

Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Control

Stability controls (Reference Control B) and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution

The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of MEDOL-10 was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls

Triplicate solutions each of the positive control and MEDOL-10 stock solutions were diluted with the Cysteine peptide stock solution so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or MEDOL-10. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls

Triplicate solutions each of the positive control and MEDOL-10 were diluted with the Lysine peptide stock solution so as to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or MEDOL-10. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation

The appearance of the MEDOL-10 and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis

The concentration of both the Cysteine and Lysine peptides in the presence of MEDOL-10 and the associated positive controls was quantified by HPLC using UV detection as detailed in the "Any other information on materials and methods incl. tables" section.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Mean depletion % of positive control was 71.9 and 57.8 for cysteine and lysine respectively.
All analytical acceptance criteria for each peptide run were met

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Overall peptide depletion value

Value:

65.6 %

At concentration:

100 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The overall depletion value of 65.6% places MEDOL-10 in the reactivity class of “high” and hence it is predicted by DPRA to be a skin sensitizer

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

31.2 %

At concentration:

100 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

100 %

At concentration:

100 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

high reactivity [in chemico]

Other effects / acceptance of results:

All analytical acceptance criteria for each peptide run were met

Solubility Assessment

The solubility of MEDOL-10 in acetonitrile at a nominal concentration of 100 mM was achieved.

Reactivity Assessment

The DPRA prediction and the reactivity of MEDOL-10 based on the overall mean and the individual depletion values in the Cysteine peptide and the Lysine peptide is presented in Table 1. Peptide calibration graphs and typical sample chromatograms are presented in Figure 1, Figure 2 and Figure 3.

All analytical acceptance criteria for each peptide run were met:

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <15%)	SD <14.9%
	Lysine	r2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r2>0.999	71.9 (SD, 0.33%, n=3)	B: 0.502 mM (CV 0.37%, n=6)	SD 0.00% (n=3)
	Lysine	r2>0.999	57.8 (SD, 0.67%, n=3)	B: 0.505 mM (CV 2.05%, n=6)	SD 0.99% (n=3)

CV Coefficient of Variation
SD Standard deviation

The depletion of peptide in the presence of MEDOL-10 was:

	Mean peak area of reference controls (μV.sec)	Mean peak area of peptide with test item (μV.sec)	Mean peptide depletion by MEDOL-10 (%)
Cysteine	Control B: 814960 (n=6)	0 (n=3)	100
Lysine	Control B: 834970 (n=6)	574740 (n=3)	31.2

 

And applying the following depletion model (below), reactivity is classed as “high reactivity” and the DPRA prediction is positive and MEDOL-10 is therefore predicted to be a skin sensitizer.

Mean of cysteine and lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

There were no co-elution peaks in either the Cysteine or Lysine assays.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Solutions of MEDOL-10 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. The overall depletion value of 65.6% places MEDOL-10 in the reactivity class of “high” and hence it is predicted by DPRA to be a skin sensitizer.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of MEDOL-10.
Solutions of MEDOL-10 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. The overall depletion value of 65.6% places MEDOL-10 in the reactivity class of “high” and hence it is predicted by DPRA to be a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1.1 CAS number: 69701-99-1
1.2 EC number: 807-159-2
1.3 Chemical name
IUPAC: (2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
Other: MEDOL-10
1.5 Structure codes
SMILES: CCC1(C)OCC(COC(=O)C=C)O1
InChI: 1/C10H16O4/c1-4-9(11)12-6-8-7-13-10(3,5-2)14-8/h4,8H,1,5-
7H2,2-3H3
Stereochemical features: Not applicable

2: General Information
2.1: Date of QPRF: 07 November 2018
2.2: Author and contact details Envigo CRS Switzerland Ltd.,Rheinstrasse 74, 4414 Füllinsdorf, Switzerland

3 Prediction
3.1 Endpoint (OECD Principle 1)
Endpoint: Skin Sensitisation (None vs Sensitiser)
Dependent variable: Classification as sensitiser or non-sensitiser

3.2 Algorithm (OECD Principle 2)
Model or submodel name: Toxicity Prediction (Extensible)Toxicity Prediction (Extensible) Skin sensitisation (None vs Sensitiser)
Model version: 4.5
Reference to QMRF: The corresponding QMRF "BIOVIA toxicity prediction model
– skin sensitiser vs nonsensitiser" is available from JRC QSAR Model Database (http://qsardb.jrc.it/qmrf) QMRF identifier (ECB Inventory): :Q50-54-55-509. The original data set was extended with 39 additional compounds from Envigo database.
Predicted values (model result): Sensitiser
Predicted values (comments): Bayesian score of 0.18 is above the best split of -1.211 indicating concordance with the predicted result. A probability of 0.75 is determinate and shows a likelihood of positive response in an experimental assay.
Input for prediction: Smiles
Descriptor values: LogP 1.056
Molecular weight (g/mol) 200.232
Number of hydrogen bond donors 0
Number of hydrogen bond acceptors 4
Number of rotatable bonds in the molecule 5
The fraction of polar surface area over the
total molecular surface area 0.197
FCFP_12: Unit functional class extended connectivity atom type fingerprint with a maximum length of 12 bonds: Not applicable

3.3 Applicability domain (OECD Principle 3)
Domains
i. All properties and OPS components are within expected ranges
ii. One unknown fingerprint feature has been found in the training set
iii. Considerations on the mechanism domain are not applicable since the contributing FCFP_12 features of the model are selected purely on their Bayesian score (statistical model)

Structural analogues (training set)
i. Methacrolein diacetate
ii. Ethylidene benzoacetate
iii. 4-(2-Nitrobutyl)-morpholine
iv. Ethylphenylglycidate
Consideration on structural analogues: With 47% the average similarity of the four analogues to the query structure is considered low. All four structures are sensitisers and thus indicating high concordance with the query structure. Accuracy between predicted and actual result is high as all four compounds are predicted correctly.
3.4: The uncertainty of the prediction (OECD principle 4)
Uncertainty may be indicated due to low similarity of structural similar compounds and one fingerprint feature not found in the training set.
3.5: The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)
Not applicable since statistical model

4 Adequacy (Optional)
4.1 Regulatory purpose: Skin sensitisation endpoint for determining property of chemical to be sensitizer or not sensitizer.
4.2: Approach for regulatory interpretation of the model result
Result is directly applicable since no conversion of the result is required.
4.3: Outcome: Sensitiser. There is uncertainty in the prediction due to low similarity of structural similar compounds and one fingerprint feature not found in the training set.
4.4 Conclusion: The predicted structure is within applicability domain, therefore this positive result may be regarded as moderate reliable.

Principles of method if other than guideline:

- Software tool(s) used including version: TOPKAT 4.5
- Justification of QSAR prediction: TOPKAT predicted MEDOL-10 to be sensitizer. According to the modelling statistics: low similarity of structural analogues and one fingerprint feature not found in the training set, the prediction may be regarded as moderate reliable.

Remarks on result:

other: Quantitative result is not available as the results are based on a QSAR prediction. Please see conclusion section for result.

1.1 CAS number: 69701-99-1
1.2 EC number: 807-159-2
1.3 Chemical name
IUPAC: (2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
Other: MEDOL-10
1.5 Structure codes
SMILES: CCC1(C)OCC(COC(=O)C=C)O1
InChI: 1/C10H16O4/c1-4-9(11)12-6-8-7-13-10(3,5-2)14-8/h4,8H,1,5-
7H2,2-3H3
Stereochemical features: Not applicable

2: General Information
2.1: Date of QPRF: 07 November 2018
2.2: Author and contact details Asta Ruzgyte Frère, PhD. Envigo CRS Switzerland Ltd.,Rheinstrasse 74, 4414 Füllinsdorf, Switzerland,Email: [e-mail]

3 Prediction
3.1 Endpoint (OECD Principle 1)
Endpoint: Skin Sensitisation (None vs Sensitiser)
Dependent variable: Classification as sensitiser or non-sensitiser

3.2 Algorithm (OECD Principle 2)
Model or submodel name: Toxicity Prediction (Extensible)Toxicity Prediction (Extensible) Skin sensitisation (None vs Sensitiser)
Model version: 4.5
Reference to QMRF: The corresponding QMRF "BIOVIA toxicity prediction model
– skin sensitiser vs nonsensitiser" is available from JRC QSAR Model Database (http://qsardb.jrc.it/qmrf) QMRF identifier (ECB Inventory): :Q50-54-55-509. The original data set was extended with 39 additional compounds from Envigo database.
Predicted values (model result): Sensitiser
Predicted values (comments): Bayesian score of 0.18 is above the best split of -1.211 indicating concordance with the predicted result. A probability of 0.75 is determinate and shows a likelihood of positive response in an experimental assay.
Input for prediction: Smiles
Descriptor values: LogP 1.056
Molecular weight (g/mol) 200.232
Number of hydrogen bond donors 0
Number of hydrogen bond acceptors 4
Number of rotatable bonds in the molecule 5
The fraction of polar surface area over the
total molecular surface area 0.197
FCFP_12: Unit functional class extended connectivity atom type fingerprint with a maximum length of 12 bonds: Not applicable

3.3 Applicability domain (OECD Principle 3)
Domains
i. All properties and OPS components are within expected ranges
ii. One unknown fingerprint feature has been found in the training set
iii. Considerations on the mechanism domain are not applicable since the contributing FCFP_12 features of the model are selected purely on their Bayesian score (statistical model)

Structural analogues (training set)
i. Methacrolein diacetate
ii. Ethylidene benzoacetate
iii. 4-(2-Nitrobutyl)-morpholine
iv. Ethylphenylglycidate
Consideration on structural analogues: With 47% the average similarity of the four analogues to the query structure is considered low. All four structures are sensitisers and thus indicating high concordance with the query structure. Accuracy between predicted and actual result is high as all four compounds are predicted correctly.
3.4: The uncertainty of the prediction (OECD principle 4)
Uncertainty may be indicated due to low similarity of structural similar compounds and one fingerprint feature not found in the training set.
3.5: The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)
Not applicable since statistical model

4 Adequacy (Optional)
4.1 Regulatory purpose: Skin sensitisation endpoint for determining property of chemical to be sensitizer or not sensitizer.
4.2: Approach for regulatory interpretation of the model result
Result is directly applicable since no conversion of the result is required.
4.3: Outcome: Sensitiser. There is uncertainty in the prediction due to low similarity of structural similar compounds and one fingerprint feature not found in the training set.
4.4 Conclusion: The predicted structure is within applicability domain, therefore this positive result may be regarded as moderate reliable.

Conclusions:

TOPKAT predicted MEDOL-10 to be sensitizer. The predicted structure is within applicability domain, therefore this positive result may be regarded as moderate reliable.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1.1 CAS number: 69701-99-1
1.2 EC number: 807-159-2
1.3 Chemical name
IUPAC: (2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
Other: MEDOL-10
1.5 Structure codes
SMILES: CCC1(C)OCC(COC(=O)C=C)O1
InChI: 1/C10H16O4/c1-4-9(11)12-6-8-7-13-10(3,5-2)14-8/h4,8H,1,5-
7H2,2-3H3
Stereochemical features: Not applicable

2: General Information
2.1: Date of QPRF: 07 November 2018
2.2: Author and contact details Envigo CRS Switzerland Ltd.,Rheinstrasse 74, 4414 Füllinsdorf, Switzerland

3 Prediction
3.1 Endpoint (OECD Principle 1)
Endpoint: Skin Sensitisation (None vs Sensitiser)
Dependent variable: Classification as sensitiser or non-sensitiser

3.2 Algorithm (OECD Principle 2)
Model or submodel name: Extension of the original CAESAR model for skin sensitisation (None vs Sensitiser) within VEGA 1.1.4
Model version: 2.1.6
Reference to QMRF: Not available
Predicted values (model result): Sensitiser
Predicted values (comments): According to VEGA's evaluation scheme the prediction may be not reliable.
Input for prediction: Smiles
Descriptor values: Not provided

3.3 Applicability domain (OECD Principle 3)
Domains
i. Predicted compound is outside applicability domain of the model.
ii. A prominent number of atom centered fragments of the compound have not been found in the compounds of the training set or are rare fragments (1 unknown fragment found: OC(O)(C)C).
iii. Considerations on the mechanism domain are not applicable since statistical model.

Structural analogues
i. Ethylene methacrylate
ii. 2-Hydroxypropyl methacrylate
iii. Diethyl maleate
iv. n-Butyl glycidyl ether
With 80% the average similarity of the four analogues to the query structure is considered high. Three out of four structures are sensitiser thus indicating moderate concordance with the query structure. Accuracy between predicted and actual result is moderate as one out of four structures is predicted as falsepositive.
3.4: The uncertainty of the prediction (OECD principle 4)
Uncertainty may be indicated due to moderate concordance, moderate accuracy, one unknown fragment in the training set. In addition, the query structure is outside applicability domain.
3.5: The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)
Not applicable since statistical model

4 Adequacy (Optional)
4.1 Regulatory purpose: Skin sensitisation endpoint for determining property of chemical to be sensitizer or not sensitizer.
4.2: Approach for regulatory interpretation of the model result
Result is directly applicable since no conversion of the result is required.
4.3: Outcome: Sensitiser. There are some uncertainty in the prediction, and compound is outside the applicability domain of the model.
4.4 Conclusion: The prediction is regarded as low reliable.

Principles of method if other than guideline:

- Software tool(s) used including version: CAESAR (VEGA 1.1.4)
- Justification of QSAR prediction: CAESAR predicted MEDOL-10 to be sensitizer. According to VEGA’s evaluation scheme, the prediction may be not reliable. The uncertainty in the prediction included moderate concordance, moderate accuracy and one unknown fragment in the training set. In addition, MEDOL-10 was outside applicability domain of the model. As the average similarity of the structural analogues is still high, the prediction may be assessed as low reliable for a weight-of-evidence approach.

Remarks on result:

other: Quantitative result is not available as the results are based on a QSAR prediction. Please see conclusion for full results.

1.1 CAS number: 69701-99-1
1.2 EC number: 807-159-2
1.3 Chemical name
IUPAC: (2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
Other: MEDOL-10
1.5 Structure codes
SMILES: CCC1(C)OCC(COC(=O)C=C)O1
InChI: 1/C10H16O4/c1-4-9(11)12-6-8-7-13-10(3,5-2)14-8/h4,8H,1,5-
7H2,2-3H3
Stereochemical features: Not applicable

2: General Information
2.1: Date of QPRF: 07 November 2018
2.2: Author and contact details Asta Ruzgyte Frère, PhD. Envigo CRS Switzerland Ltd.,Rheinstrasse 74, 4414 Füllinsdorf, Switzerland,Email: [e-mail]

3 Prediction
3.1 Endpoint (OECD Principle 1)
Endpoint: Skin Sensitisation (None vs Sensitiser)
Dependent variable: Classification as sensitiser or non-sensitiser

3.2 Algorithm (OECD Principle 2)
Model or submodel name: Extension of the original CAESAR model for skin sensitisation (None vs Sensitiser) within VEGA 1.1.4
Model version: 2.1.6
Reference to QMRF: Not available
Predicted values (model result): Sensitiser
Predicted values (comments): According to VEGA's evaluation scheme the prediction may be not reliable.
Input for prediction: Smiles
Descriptor values: Not provided

3.3 Applicability domain (OECD Principle 3)
Domains
i. Predicted compound is outside applicability domain of the model.
ii. A prominent number of atom centered fragments of the compound have not been found in the compounds of the training set or are rare fragments (1 unknown fragment found: OC(O)(C)C).
iii. Considerations on the mechanism domain are not applicable since statistical model.

Structural analogues
i. Ethylene methacrylate
ii. 2-Hydroxypropyl methacrylate
iii. Diethyl maleate
iv. n-Butyl glycidyl ether
With 80% the average similarity of the four analogues to the query structure is considered high. Three out of four structures are sensitiser thus indicating moderate concordance with the query structure. Accuracy between predicted and actual result is moderate as one out of four structures is predicted as falsepositive.
3.4: The uncertainty of the prediction (OECD principle 4)
Uncertainty may be indicated due to moderate concordance, moderate accuracy, one unknown fragment in the training set. In addition, the query structure is outside applicability domain.
3.5: The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5)
Not applicable since statistical model

4 Adequacy (Optional)
4.1 Regulatory purpose: Skin sensitisation endpoint for determining property of chemical to be sensitizer or not sensitizer.
4.2: Approach for regulatory interpretation of the model result
Result is directly applicable since no conversion of the result is required.
4.3: Outcome: Sensitiser. There are some uncertainty in the prediction, and compound is outside the applicability domain of the model.
4.4 Conclusion: The prediction is regarded as low reliable.

Conclusions:

CAESAR predicted MEDOL-10 to be sensitizer. According to VEGA’s evaluation scheme, the
prediction may be not reliable. The uncertainty in the prediction included moderate
concordance, moderate accuracy and one unknown fragment in the training set. In addition,
MEDOL-10 was outside applicability domain of the model. As the average similarity of the
structural analogues is still high, the prediction may be assessed as low reliable for a weight of-
evidence approach.

Reference 5

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

1. Derek Nexus

2. Derek Nexus: 5.0.2, Nexus: 2.1.1

3. SMILES: O=C(OCC1OC(OC1)(C)CC)C=C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Derek Nexus is expert, knowledge-based toxicology software and has been designed to meet the OECD validation principles
- Defined endpoint: Skin sensitization
- Unambiguous algorithm: for details please refer to https://www.lhasalimited.org/
- Defined domain of applicability: No misclassified or unclassified features.
- Appropriate measures of goodness-of-fit and robustness and predictivity: N/A
- Mechanistic interpretation:Skin sensitisation: murine local lymph node assay (LLNA), guinea pig maximisation test (GPMT), guinea pig single injection adjuvant test (SIAT) Potential mechanism (alpha,beta-unsaturated ester): Hapten acting as an electrophilic Michael acceptor [Aptula and Roberts].

This alert describes the skin sensitisation of alpha,beta-unsaturated esters and precursors which interact with skin proteins via a Michael addition mechanism [Ashby et al]. By analogy with beta-disubstituted alpha,beta-unsaturated ketones, beta-disubstituted alpha,beta-unsaturated esters are less susceptible to Michael addition and are therefore excluded from the scope of the current alert.

A number of chemicals in a series of alpha,beta-unsaturated ester precursors have been shown to give a positive response in a modified single injection adjuvant test in the guinea pig [Franot et al 1994a, Franot et al 1994b]. Human skin sensitisation has also been demonstrated in the maximisation test for diethyl maleate [Opdyke]. Mechanistic studies suggest that reaction with nucleophilic skin protein groups predominantly involves elimination followed by Michael addition rather than a single-step substitution reaction, although the latter may also contribute in part. Several of the same chemicals have also been examined in the local lymph node assay in the mouse, generally yielding negative responses [Ashby et al].

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain: N/A
- Structural domain: N/A
- Mechanistic domain: N/A
- Similarity with analogues in the training set: N/A
- Other considerations (as appropriate): no measure of applicability domain in DEREK.

6. ADEQUACY OF THE RESULT
Sufficient for use as part of a weight of evidence.

Principles of method if other than guideline:

- Software tool(s) used including version: Derek Nexus: 5.0.2.
- Model(s) used: Nexus: 2.1.1
- Model description: See field 'Justification for non-standard information'.
- Justification of QSAR prediction: See field 'Justification for type of information'.

Key result

Group:

other:

Parameter:

other:

Remarks on result:

positive indication of skin sensitisation

Remarks:

Skin sensitisation in mammal is PLAUSIBLE Alert matched: 481 alpha,beta-Unsaturated ester or precursor

Skin sensitisation in mammal is PLAUSIBLE
Alert matched # 481 alpha,beta-Unsaturated ester or precursor

EC3 Result for Derek EC3 Model - 1.0.6

Species: mammal

Alert: 481 alpha,beta-Unsaturated ester or precursor
Dataset Name: Derek EC3 Model - 1.0.6
Dataset Certified: Yes
Predicted LLNA EC3: 0.44% (strong sensitiser)
Experimental Match: No exact match found
Compounds used in calculation: 10/25

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Sufficient for use as part of weight of evidence.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

MEDOL-10 may be a weak skin sensitizer (Category 1A)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation.OECD 442C: Solutions of MEDOL-10 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. The overall depletion value of 65.6% places MEDOL-10 in the reactivity class of “high” and hence it is predicted by DPRA to be a skin sensitizer.

Skin sensitisation.OECD 442E: The test item MEDOL-10 with a log Pow of 1.92 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Skin sensitisation.QSAR TOPKAT: TOPKAT predicted MEDOL-10 to be sensitizer. The predicted structure is within applicability domain, therefore this positive result may be regarded as moderate reliable.

Skin sensitisation.QSAR CAESAR: CAESAR predicted MEDOL-10 to be sensitizer. According to VEGA’s evaluation scheme, the prediction may be not reliable. The uncertainty in the prediction included moderate concordance, moderate accuracy and one unknown fragment in the training set. In addition, MEDOL-10 was outside applicability domain of the model. As the average similarity of the structural analogues is still high, the prediction may be assessed as low reliable for a weight of- evidence approach.

The read-across assessment was performed with the OECD QSAR Toolbox taking into account the qualitative and quantitative approach. In qualitative read-across approach, MEDOL-10 was predicted as a skin sensitizer with low confidence (p-value: 0.5). The prediction was based on five structural analogue values (structural similarity 50-60%), there three of them were skin sensitizer. In quantitative read-across approach, MEDOL-10 was predicted with LLNA EC3 value of 12.9% using the conservative data usage approach of the lower median value of the five structural analogues (structural similarity 40-60%).

In conclusion, the skin metabolites/transformation products of MEDOL-10 are not likely to be sensitizers, therefore pre- and pro-haptens are not expected. Taking into account the assessed computational tools, their reliability and the mechanistic details there is likelihood, that unchanged MEDOL-10 may be a weak skin sensitizer.

Skin sensitisation.QSAR DEREK: DEREK predicted MEDOL-10 to be sensitizer with high reliability, and predicted an EC3 of 0.44%, suggesting the compound is a strong strong sensitiser.

As there was conflicting evidence for classification. The lowest value was used for classification and risk assessment. thus the compound Medol-10 was concluded to be a Category 1A skin sensitiser.